Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Proposed hypothesis of GSK-3 ß inhibition for stimulating Wnt/ß-catenin signaling pathway which triggers liver regeneration process.

Almost every human organ has a poor ability to regenerate, notable exceptions are liver, skin, gut, etc. Molecular and cellular underpinnings of liver regeneration might pave the way for novel treatments concerned with chronic liver disorder. Such treatments would eliminate the disadvantages of liver transplantation, such as a scarcity of donor organs, a lengthy waitlist, significant medical expenses, surgical complications, and the necessity for lifelong immunosuppressive medications. Advancement in the development of regenerative therapy is giving hope to those suffering from end-stage liver disorder. The regeneration process is unique, intricate, and well coordinated, which involve the interaction of numerous signaling pathways, cytokines, and growth factor. Various signaling pathways for liver regeneration are HO-1/BER pathway, Tweak/Fn14 signaling pathway, Hippo pathway, Wnt/beta-catenin pathway, Hedgehog signaling pathway, bile acids repairing pathway, serotonin (5HT) pathway, estrogen pathway, thyrotropin-releasing hormone (TRH) pathway, insulin repairing pathway, etc. The in vitro scientific literature revealed that numerous GSK-3 ß inhibitors (LY 2090314, AR-A014418, Tideglusib, Solasodine, CHIR99021, 9-ING-41, SB-216763) play an important role in stimulating the liver regeneration process. Similarly, from the above discussion, the direction is highlighted to emphasize the proposed molecular Wnt/ß-catenin signaling pathway which is associated with GSK-3 ß inhibition for the induction of the repairing and regeneration process.

Shear stress-induced cellular senescence blunts liver regeneration through Notch-sirtuin 1-P21/P16 axis.

BACKGROUND AND AIMS: The mechanisms involved in liver regeneration after partial hepatectomy (pHx) are complicated. Cellular senescence, once linked to aging, plays a pivotal role in wound repair. However, the regulatory effects of cellular senescence on liver regeneration have not been fully elucidated. APPROACH AND RESULTS: Mice subjected to pHx were analyzed 14 days after surgery. The incomplete remodeling of liver sinusoids affected shear stress-induced endothelial nitric oxide synthase (eNOS) signaling on day 14, resulting in the accumulation of senescent LSECs. Removing macrophages to augment LSEC senescence led to a malfunction of the regenerating liver. A dynamic fluctuation in Notch activity accompanied senescent LSEC accumulation during liver regeneration. Endothelial Notch activation by using Cdh5-CreERT NICeCA mice triggered LSEC senescence and senescence-associated secretory phenotype, which disrupted liver regeneration. Blocking the Notch by γ-secretase inhibitor (GSI) diminished senescence and promoted LSEC expansion. Mechanically, Notch-hairy and enhancer of split 1 signaling inhibited sirtuin 1 (Sirt1) transcription by binding to its promoter region. Activation of Sirt1 by SRT1720 neutralized the up-regulation of P53, P21, and P16 caused by Notch activation and eliminated Notch-driven LSEC senescence. Finally, Sirt1 activator promoted liver regeneration by abrogating LSEC senescence and improving sinusoid remodeling. CONCLUSIONS: Shear stress-induced LSEC senescence driven by Notch interferes with liver regeneration after pHx. Sirt1 inhibition accelerates liver regeneration by abrogating Notch-driven senescence, providing a potential opportunity to target senescent cells and facilitate liver repair after injury.

Pterostilbene alleviates liver ischemia/reperfusion injury via PINK1-mediated mitophagy.

Hepatic ischemia/reperfusion (I/R) injury contributes to morbidity and mortality during liver resection or transplantation, with limited effective treatments available. Here, we investigated the potential benefits and underlying mechanisms of pterostilbene (Pt), a natural component of blueberries and grapes, in preventing hepatic I/R injury. Male C57BL/6 mice subjected to partial warm hepatic I/R and human hepatocyte cell line L02 cells exposed to anoxia/reoxygenation (A/R) were used as in vivo and in vitro models, respectively. Our findings showed that pretreatment with Pt ameliorated hepatic I/R injury by improving liver histology, decreasing hepatocyte apoptosis, and reducing plasma ALT and AST levels. Likewise, cell apoptosis, mitochondrial membrane dysfunction, and mitochondrial ROS overproduction in L02 cells triggered by the A/R challenge in vitro were reduced due to Pt administration. Mechanistically, Pt treatment efficiently enhanced mitophagy and upregulated PINK1, Parkin, and LC3B expression. Notably, the protective effect of Pt was largely abrogated after cells were transfected with PINK1 siRNA. Moreover, Pt pretreatment promoted hepatocyte proliferation and liver regeneration in the late phase of hepatic I/R. In conclusion, our findings provide evidence that Pt exerts hepatoprotective effects in hepatic I/R injury by upregulating PINK1-mediated mitophagy.

YAP-TEAD mediates PPAR α-induced hepatomegaly and liver regeneration in mice.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor α (PPARα, NR1C1) is a ligand-activated nuclear receptor involved in the regulation of lipid catabolism and energy homeostasis. PPARα activation induces hepatomegaly and plays an important role in liver regeneration, but the underlying mechanisms remain unclear. APPROACH AND RESULTS: In this study, the effect of PPARα activation on liver enlargement and regeneration was investigated in several strains of genetically modified mice. PPARα activation by the specific agonist WY-14643 significantly induced hepatomegaly and accelerated liver regeneration after 70% partial hepatectomy (PHx) in wild-type mice and Pparafl/fl mice, while these effects were abolished in hepatocyte-specific Ppara-deficient (PparaΔHep ) mice. Moreover, PPARα activation promoted hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. Mechanistically, PPARα activation regulated expression of yes-associated protein (YAP) and its downstream targets (connective tissue growth factor, cysteine-rich angiogenic inducer 61, and ankyrin repeat domain 1) as well as proliferation-related proteins (cyclins A1, D1, and E1). Binding of YAP with the PPARα E domain was critical for the interaction between YAP and PPARα. PPARα activation further induced nuclear translocation of YAP. Disruption of the YAP-transcriptional enhancer factor domain family member (TEAD) association significantly suppressed PPARα-induced hepatomegaly and hepatocyte enlargement and proliferation. In addition, PPARα failed to induce hepatomegaly in adeno-associated virus-Yap short hairpin RNA-treated mice and liver-specific Yap-deficient mice. Blockade of YAP signaling abolished PPARα-induced hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. CONCLUSIONS: This study revealed a function of PPARα in regulating liver size and liver regeneration through activation of the YAP-TEAD signaling pathway. These findings have implications for understanding the physiological functions of PPARα and suggest its potential for manipulation of liver size and liver regeneration.

Liver regeneration after extensive hepatectomy in rats: effect of preoperative chemotherapy with intravenous 5-fluorouracil

Purpose: To evaluate the effect of preoperative intravenous chemotherapy with 5-fluorouracil on liver regeneration in an experimental model of major hepatectomy in rats. Methods: Wistar rats were divided into two groups of 20 animals each and submitted to 70% hepatectomy 24 h after intravenous injection of 5-fluorouracil 20 mg/kg (fluorouracil group, FG) or 0.9% saline (control group, CG). After hepatectomy, each group was subdivided into two subgroups of 10 animals each according to the day of sacrifice (24 h or 7 days). Liver weight during regeneration, liver regeneration rate using Kwon formula, and the immunohistochemical markers proliferating cell nuclear antigen (PCNA) and Ki-67 were used to assess liver regeneration. Results: At early phase (24 h after hepatectomy) it was demonstrated the negative effect of 5-fluorouracil on liver regeneration when assessed by Kwon formula (p < 0.0001), PCNA analysis (p = 0.02). With regeneration process complete (7 days), it was possible to demonstrate the sustained impairment of chemotherapy with 5-fluorouracil on hepatocytes regeneration phenomenon when measured by Kwon formula (p = 0.009), PCNA analysis (p = 0.0001) and Ki-67 analysis (0.001). Conclusions: Preoperative chemotherapy with intravenous 5-fluorouracil negatively affected the mechanisms of liver regeneration after major hepatectomy in rats.

Evaluation of Mesenchymal Stem Cells with Conditioned Media and m-EGF for Regeneration of Liver Tissue After Partial Hepatectomy in Wistar Rats.

BACKGROUND/AIMS: Liver is considered as the vital organ in the body as it performs various essential functions. Following an injury to the liver, the repair process even though initially beneficial becomes pathogenic when it is not controlled appropriately. Extensive accumulation of extracellular matrix (ECM) components can ultimately lead to cirrhosis and liver failure. Thus, the ideal strategy to treat a liver injury is to generate new hepatocytes replacing damaged cells without causing excessive ECM deposition. The objective of this study was to evaluate the potential of mesenchymal stem cells, conditioned media and murine epidermal growth factor (m-EGF) in liver regeneration following partial hepatectomy in a rat model. METHODS: The animals were anaesthetized and a midline laparotomy was done. The liver was exposed and the left lateral and median lobes were ligated and resected out (about 65-70% of total liver mass). The muscles and skin were sutured in routine fashion and thus the rat model of partial hepatectomy was prepared. The animal models were equally distributed into 4 different groups namely A, B, C and D and treated with PBS, conditioned media, mesenchymal stem cells and epidermal growth factor respectively. The liver regeneration was assessed based on clinical, haemato-biochemical, colour imaging, histopathological and immune-histochemical parameters. RESULTS: Partial hepatectomy model with surgical removal of 65-70% liver lobe was standardized and successfully used in this study. Alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), bilirubin, transaminases were significantly higher (P<0.05) in group A indicating that the liver damage was not restored properly. Colour digital imaging, histopathological and immune-histochemistry observations revealed that a better liver regeneration was observed in groups C and D, followed by groups B and A. Regeneration coefficient calculated based on liver weight was higher in groups C and D as compared to group A. CONCLUSION: Rat bone marrow-derived mesenchymal stem cells were found to induce hepatocytes proliferation; whereas EGF induced more angiogenesis. Conditioned media was not as effective as stem cells and EGF in liver tissue repair.

Alleviation of CCCP-induced mitochondrial injury by augmenter of liver regeneration via the PINK1/Parkin pathway-dependent mitophagy.

The occurrence of liver diseases is attributed to mitochondrial damage. Mitophagy selectively removes dysfunctional mitochondria, thereby preserving mitochondrial function. Augmenter of liver regeneration (ALR) protects the mitochondria from injury. However, whether ALR protection is associated with mitophagy remains unclear. In this study, mitochondrial damage was induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and long-form ALR (lfRNA)-mediated protection against this damage was investigated. Treatment of HepG2 cells with CCCP elevated the level of intracellular ROS, inhibited ATP production, and increased the mitochondrial membrane potential and cell apoptotic rate. However, in lfALR-transfected cells, CCCP-induced cell injury was clearly alleviated, the apoptosis and ROS levels clearly declined, and the ATP production was significantly enhanced as compared with that in vector-Tx cells. Furthermore, lfALR overexpression promoted autophagy and mitophagy via a PINK1/Parkin-dependent pathway, whereas knockdown of ALR suppressed mitophagy. In lfALR-transfected cells, the phosphorylation of AKT was decreased, thus, downregulating the phosphorylation of the transcription factor FOXO3a at Ser315. In contrast, the phosphorylation of AMPK was enhanced, thereby upregulating the phosphorylation of FOXO3a at Ser413. Consequently, FOXO3a's nuclear translocation and binding to the promoter region of PINK1 was enhanced, and the accumulation of PINK1/Parkin in mitochondria increased. Meanwhile, short-form ALR (sfALR) also increased PINK1 expression through FOXO3a with the similar pathway to lfALR. In conclusion, our data suggest a novel mechanism through which both lfALR and sfALR protect mitochondria by promoting PINK1/Parkin-dependent mitophagy through FOXO3a activation.

Vanillin augments liver regeneration effectively in Thioacetamide induced liver fibrosis rat model.

AIMS: This study has been designed to investigate the role of vanillin either as prophylaxis or treatment in liver regeneration augmentation and liver fibrosis regression in thioacetamide (TAA) induced liver damage. MATERIALS AND METHODS: Animals were injected with TAA to induce liver injury (200mg/kg twice weekly) for 8 weeks. In vanillin prophylaxis group; rats were administered vanillin (100 mg/Kg; IP, daily) from day 1 of TAA injection for 8 weeks. In vanillin treatment group; rats were confronted with the same dose of TAA injection for 8 weeks then treated with vanillin (100 mg/Kg, IP, daily) for 4 weeks. ALT, AST activities, serum albumin, hepatic GSH, MDA, HGF, VEGF, IL-6 and TNF-α levels were measured and also, MMP-2, TIMP-1 and cyclin D gene expression were determined. Liver sections were stained with H&E and Sirius red and immunostained for Ki-67 and α-SMA for histological and immunohistological changes analysis. KEY FINDINGS: Vanillin improved liver function and histology. Also, showed a remarkable increase in hepatic HGF and VEGF level, and up-regulation of cyclin D1 expression accompanied by a significant up-regulation of MMP-2 and down- regulation of TIMP-1. All these effects were accompanied by TNF-α, IL-6 and oxidative stress significant attenuation. SIGNIFICANCE: In conclusion, vanillin enhanced liver regeneration in TAA induced liver damage model; targeting growth factors (HGF, VEGF) and cellular proliferation marker cyclin D1. As well as stimulating fibrosis regression by inhibition of ECM accumulation and enhancing its degradation.

Perioperative rifaximin is not associated with enhanced functional and volumetric recovery after major liver resection.

The objective of this randomized controlled trial (RCT) was to assess the impact of rifaximin on the course of liver function, liver regeneration and volumetric recovery in patients undergoing major hepatectomy. The ARROW trial was an investigator initiated, single-center, open-label, phase 3 RCT with two parallel treatment groups, conducted at our hepatobiliary center from 03/2016 to 07/2020. Patients undergoing major hepatectomy were eligible and randomly assigned 1:1 to receive oral rifaximin (550 mg twice daily for 7-10 or 14-21 days in case of portal vein embolization preoperatively and 7 days postoperatively) versus no intervention. Primary endpoint was the relative increase in postoperative liver function measured by LiMAx from postoperative day (POD) 4 to 7. Secondary endpoint were the course of liver function and liver volume during the study period as well as postoperative morbidity and mortality. Between 2016 and 2020, 45 patients were randomized and 35 patients (16 individuals in the rifaximin and 19 individuals in the control group) were eligible for per-protocol analysis. The study was prematurely terminated following interim analysis, due to the unlikelihood of reaching a significant primary endpoint. The median relative increase in liver function from POD 4 to POD 7 was 27% in the rifaximin group and 41% in the control group (p = 0.399). Further, no significant difference was found in terms of any other endpoints of functional liver- and volume regeneration or perioperative surgical complications following the application of rifaximin versus no intervention. Perioperative application of rifaximin has no effect on functional or volumetric regeneration after major hepatectomy (NCT02555293; EudraCT 2013-004644-28).

Selective HIF stabilization alleviates hepatocellular steatosis and ballooning in a rodent model of 70% liver resection.

BACKGROUND: Small-for-size syndrome (SFSS) looms over patients needing liver resection or living-donor transplantation. Hypoxia has been shown to be crucial for the successful outcome of liver resection in the very early postoperative phase. While poorly acceptable as such in real-world clinical practice, hypoxia responses can still be simulated by pharmacologically raising levels of its transducers, the hypoxia-inducible factors (HIFs). We aimed to assess the potential role of a selective inhibitor of HIF degradation in 70% hepatectomy (70%Hx). METHODS: In a pilot study, we tested the required dose of roxadustat to stabilize liver HIF1α. We then performed 70%Hx in 8-week-old male Lewis rats and administered 25 mg/kg of roxadustat (RXD25) at the end of the procedure. Regeneration was assessed: ki67 and 5-ethynyl-2'-deoxyuridine (EdU) immunofluorescent labeling, and histological parameters. We also assessed liver function via a blood panel and functional gadoxetate-enhanced magnetic resonance imaging (MRI), up to 47 h after the procedure. Metabolic results were analyzed by means of RNA sequencing (RNAseq). RESULTS: Roxadustat effectively increased early HIF1α transactivity. Liver function did not appear to be improved nor liver regeneration to be accelerated by the experimental compound. However, treated livers showed a mitigation in hepatocellular steatosis and ballooning, known markers of cellular stress after liver resection. RNAseq confirmed that roxadustat unexpectedly increases lipid breakdown and cellular respiration. CONCLUSIONS: Selective HIF stabilization did not result in an enhanced liver function after standard liver resection, but it induced interesting metabolic changes that are worth studying for their possible role in extended liver resections and fatty liver diseases.

Hepatic NF-κB-Inducing Kinase and Inhibitor of NF-κB Kinase Subunit α Promote Liver Oxidative Stress, Ferroptosis, and Liver Injury.

Drug-induced hepatotoxicity limits development of new effective medications. Drugs and numerous endogenous/exogenous agents are metabolized/detoxified by hepatocytes, during which reactive oxygen species (ROS) are generated as a by-product. ROS has broad adverse effects on liver function and integrity, including damaging hepatocyte proteins, lipids, and DNA and promoting liver inflammation and fibrosis. ROS in concert with iron overload drives ferroptosis. Hepatic nuclear factor kappa B (NF-κB)-inducing kinase (NIK) is aberrantly activated in a broad spectrum of liver disease. NIK phosphorylates and activates inhibitor of NF-κB kinase subunit alpha (IKKα), and the hepatic NIK/IKKα cascade suppresses liver regeneration. However, the NIK/IKKα pathway has not been explored in drug-induced liver injury. Here, we identify hepatic NIK as a previously unrecognized mediator for acetaminophen (APAP)-induced acute liver failure. APAP treatment increased both NIK transcription and NIK protein stability in primary hepatocytes as well as in liver in mice. Hepatocyte-specific overexpression of NIK augmented APAP-induced liver oxidative stress in mice and increased hepatocyte death and mortality in a ROS-dependent manner. Conversely, hepatocyte-specific ablation of NIK or IKKα mitigated APAP-elicited hepatotoxicity and mortality. NIK increased lipid peroxidation and cell death in APAP-stimulated primary hepatocytes. Pretreatment with antioxidants or ferroptosis inhibitors blocked NIK/APAP-induced hepatocyte death. Conclusion: We unravel a previously unrecognized NIK/IKKα/ROS/ferroptosis axis engaged in liver disease progression.

Molecular mechanisms and therapeutic implications of dihydromyricetin in liver disease.

Recent studies demonstrated that dihydromyricetin (DHM) has prominent therapeutic effects on liver injury and liver cancer. By summarizing the current preclinical in vitro and in vivo studies, the present review examines the preventive and therapeutic effects of DHM on liver disorders as well as its potential mechanisms. Briefly, in both chemical- and alcohol-induced liver injury models, DHM ameliorates hepatocyte necrosis and steatosis while promoting liver regeneration. In addition, DHM can alleviate nonalcoholic fatty liver disease (NAFLD) via regulating lipid/glucose metabolism, probably due to its anti-inflammatory or sirtuins-dependent mechanisms. Furthermore, DHM treatment inhibits cell proliferation, induces apoptosis and autophagy and regulates redox balance in liver cancer cells, thus exhibiting remarkable anti-cancer effects. The pharmacological mechanisms of DHM may be associated with its anti-inflammatory, anti-oxidative and apoptosis-regulatory benefits. With the accumulating interests in utilizing natural products to target common diseases, our work aims to improve the understanding of DHM acting as a novel drug candidate for liver diseases and to accelerate its translation from bench to bedside.

Deciphering the Dynamic Complexities of the Liver Microenvironment - Toward a Better Understanding of Immune-Mediated liver Injury Caused by Immune Checkpoint Inhibitors (ILICI).

Immune checkpoint inhibitors (ICIs) represent a promising therapy for many types of cancer. However, only a portion of patients respond to this therapy and some patients develop clinically significant immune-mediated liver injury caused by immune checkpoint inhibitors (ILICI), an immune-related adverse event (irAE) that may require the interruption or termination of treatment and administration of systemic corticosteroids or other immunosuppressive agents. Although the incidence of ILICI is lower with monotherapy, the surge in combining ICIs with chemotherapy, targeted therapy, and combination of different ICIs has led to an increase in the incidence and severity of ILICI - a major challenge for development of effective and safe ICI therapy. In this review, we highlight the importance and contribution of the liver microenvironment to ILICI by focusing on the emerging roles of resident liver cells in modulating immune homeostasis and hepatocyte regeneration, two important decisive factors that dictate the initiation, progression, and recovery from ILICI. Based on the proposed contribution of the liver microenvironment on ICILI, we discuss the clinical characteristics of ILICI in patients with preexisting liver diseases, as well as the challenges of identifying prognostic biomarkers to guide the clinical management of severe ILICI. A better understanding of the liver microenvironment may lead to novel strategies and identification of novel biomarkers for effective management of ILICI.

Resolution of fibrosis by siRNA HSP47 in vitamin A-coupled liposomes induces regeneration of chronically injured livers.

BACKGROUND AND AIM: In chronic hepatic diseases where treatment strategies are not available, deposited fibrotic tissues deteriorate the intrinsic regeneration capacity of the liver by creating special restrictions. Thus, if the anti-fibrosis modality is efficiently applied, the regeneration capacity of the liver should be reactivated even in such refractory hepatic diseases. METHODS: Rat liver fibrosis was induced by dimethyl-nitrosamine (DMN). Another liver fibrosis model was established in CCl4 treated Sox9CreERT2ROSA26: YFP mice. To resolve hepatic fibrosis, vitamin A-coupled liposomes containing siRNA HSP47 (VA-liposome siHSP47) were employed. EpCAM + hepatic progenitor cells from GFP rats were transplanted to DMN rat liver to examine their trans-differentiation into hepatic cells after resolution of liver fibrosis. RESULTS: Even under continuous exposure to such strong hepatotoxin as DMN, rats undergoing VA-liposome siHSP47 treatment showed an increment of DNA synthesis of hepatocytes with the concomitant restoration of impaired liver weight and normalization of albumin levels. These results were consistent with the observation that GFP + EpCAM hepatic progenitor cells transplanted to DMN rat liver, trans-differentiated into GFP + mature hepatic cells after VA-liposome siHSP47 treatment. Another rodent model also proved regeneration potential of the fibrotic liver in CCl4 administered Sox9CreERT2ROSA26: YFP mice, VA-liposome siHSP47 treatment-induced restoration of liver weight and trans-differentiation of YEP + Sox9 + cells into YFP + hepatic cells, although because of relatively mild hepatotoxicity of CCl4, undamaged hepatocytes also proliferated. CONCLUSIONS: These results demonstrated that regeneration of chronically damaged liver indeed occurs after anti-fibrosis treatment even under continuous exposure to hepatotoxin, which promises a significant benefit of the anti-fibrosis therapy for refractory liver diseases.

Astragaloside IV Suppresses Hepatic Proliferation in Regenerating Rat Liver after 70% Partial Hepatectomy via Down-Regulation of Cell Cycle Pathway and DNA Replication.

Astragaloside IV (AS-IV) is one of the major bio-active ingredients of huang qi which is the dried root of Astragalus membranaceus (a traditional Chinese medicinal plant). The pharmacological effects of AS-IV, including anti-oxidative, anti-cancer, and anti-diabetic effects have been actively studied, however, the effects of AS-IV on liver regeneration have not yet been fully described. Thus, the aim of this study was to explore the effects of AS-IV on regenerating liver after 70% partial hepatectomy (PHx) in rats. Differentially expressed mRNAs, proliferative marker and growth factors were analyzed. AS-IV (10 mg/kg) was administrated orally 2 h before surgery. We found 20 core genes showed effects of AS-IV, many of which were involved with functions related to DNA replication during cell division. AS-IV down-regulates MAPK signaling, PI3/Akt signaling, and cell cycle pathway. Hepatocyte growth factor (HGF) and cyclin D1 expression were also decreased by AS-IV administration. Transforming growth factor ß1 (TGFß1, growth regulation signal) was slightly increased. In short, AS-IV down-regulated proliferative signals and genes related to DNA replication. In conclusion, AS-IV showed anti-proliferative activity in regenerating liver tissue after 70% PHx.

Macrophage potentiates the recovery of liver zonation and metabolic function after acute liver injury.

The liver is an exclusive organ with tremendous regenerative capacity. Liver metabolic functions exhibit spatial heterogeneity, reflecting liver zonation. The mechanisms controlling the proliferation of hepatocytes and the accompanying matrix reconstruction during regeneration have been well explored, but the recovery potential of differentiated metabolic functions and zonation after liver injury remains unclear. We employed a mouse model of carbon tetrachloride (CCl4) induced-acute liver injury with clodronate-induced macrophage depletion to clarify the impact of liver injury on liver metabolism and recovery dynamics of metabolic function and liver zonation during regeneration. Depleting macrophages suppressed tissue remodelling and partially delayed cell proliferation during regeneration after liver injury. In addition, recovery of metabolic functions was delayed by suppressing the tissue remodelling caused by the depleted macrophages. The model revealed that drug metabolic function was resilient against the dysfunction caused by liver injury, but glutamine synthesis was not. Metabolomic analysis revealed that liver branched-chain amino acid (BCAA) and carbohydrate metabolism were suppressed by injury. The plasma BCAA concentration reflected recovery of hepatic function during regeneration. Our study reveals one aspect of the regenerative machinery for hepatic metabolism following acute liver injury.

Design and synthesis of 1H-pyrazolo[3,4-b]pyridines targeting mitogen-activated protein kinase kinase 4 (MKK4) - A promising target for liver regeneration.

Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a major regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this protein kinase. Starting from the approved BRAFV600E inhibitor vemurafenib (8), that showed a high off-target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4 could be conserved while the selectivity against off-target protein kinases was slightly improved. Further modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7, JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective MKK4 inhibitors in this class.

The combined induction of liver progenitor cells and the suppression of stellate cells by small molecules reverts chronic hepatic dysfunction.

Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis. Methods:In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24+ liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24+ cells compared with CCL4-induced CD24+ cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24+ cells in human hepatocyte-spheroids from fibrotic liver tissues. Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24+ progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24+ cells induced by CCL4 alone, HACY-induced CD24+ cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24+ cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro. Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24+ progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.

Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.

BACKGROUND AND AIMS: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration. APPROACH AND RESULTS: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy. Using translational noninvasive imaging techniques, CDDO-Me was shown to enhance the rate of restoration of liver volume (MRI) and improve liver function (multispectral optoacoustic imaging of indocyanine green clearance) in wild-type, but not Nrf2 null, mice following partial hepatectomy. Using immunofluorescence imaging and whole transcriptome analysis, these effects were found to be associated with an increase in hepatocyte hypertrophy and proliferation, the suppression of immune and inflammatory signals, and metabolic adaptation in the remnant liver tissue. Similar processes were modulated following exposure of primary human hepatocytes to CDDO-Me, highlighting the potential relevance of our findings to patients. CONCLUSIONS: Our results indicate that pharmacological activation of Nrf2 is a promising strategy for enhancing functional liver regeneration. Such an approach could therefore aid the recovery of patients undergoing liver surgery and support the treatment of acute and chronic liver disease.